Blood purification

ABSTRACT

Extracorporeal apparatus for selective removal of pathogenic factors, i.e. antigens, from blood by circulating the blood through hollow fibers which, exterior to the lumen are in proximity to antibodies, i.e. proteins having strong biospecific activity for the pathogenic factors. In some situations, the antibody is segregated in a liquid medium outside the hollow fiber to improve mass transfer and the antigen penetrates the ultrafilter wall of the fiber to join the antibody.

RELATED APPLICATION

This invention is a divisional of U.S. patent application Ser. No.711,304 (now U.S. Pat. No. 4,714,556) filed on Mar. 13, 1985 by Clara M.Ambrus and Csaba G. Horvath and entitled BLOOD PURIFICATION.

Ser. No. 711,304 was a continuation-in-part of U.S. patent applicationSer. No. 406,495 filed on Aug. 9, 1982, and a continuation-in-part ofSer. No. 650,772 filed on Sept. 13, 1984 which was itself acontinuation-in-part of Ser. No. 473,814, filed on Mar. 9, 1983, whichwas itself a continuation-in-part of Ser. No. 278,631 filed on June 29,1981 by Clara M. Ambrus and Csaba G. Horvath. Ser. No. 650,772 is nowU.S. Pat. No. 4,612,122. Ser. Nos. 406,495, 473,814 and 278,631 are nowabandoned.

BACKGROUND OF THE INVENTION

The present invention relates to the removal of pathogenic substancesfrom blood.

Earlier work includes the treatment of blood to remove pathogenicmaterial by a variety of physical means including, for example, dialysistechniques and the like. Hemodialysis, however is not a sufficientlyspecific procedure. Also if one removes smaller molecules such assalicylates or barbiturates by hemodialysis, the procedure is slow andincomplete because it depends upon the concentration of the poison inthe blood plasma. Larger molecules, such as proteins, cannot be removedby dialysis, but only by plasmapheresis and exchange-transfusionprocedures. Such procedures remove more of the blood than is actuallyrequired. Thus, in plasmapheresis, there is need for replacement fluid.In exchange transfusion, large amounts of blood are needed, and thereremains the problem of dealing with antigens such as hepatitis virusfrom the transfused blood.

More recently, centrifugation of the circulating blood plasma andperfusion over an immunoabsorbent bed has been utilized, underexperimental conditions, for the removal of antibodies by Protein A incancer patients. This procedure requires a complex apparatus for theseparation of plasma by centrifugation, separate passage of plasma overthe absorbing bed, and subsequent reconsitution with the cellularelements of blood before returning the blood to the patient. Thecomplexity of the process gives rise to errors, and limits its use tospecific centers having equipment and necessary highly-trainedtechnicians to operate the equipment. Also there is an undesirable riskof loss of blood components, of hemolysis, or other untoward reactions,particularly since a relatively high fraction of the patients' totalblood volume is outside the body during the procedure.

In hindsight, review of other prior art now known to the inventors, itis noted that there are numerous membrane-moderated separation devicesknown in the art. For example, U.S. Pat. No. 4,375,414 to Strahilevitzdiscloses immobilizing immunoactive materials on the side of a membraneon which the fluid being treated is flowing or having the materialcarried in fluid on the opposite side of the membrane.

Breslau et al, in U.S. Pat. No. 4,266,026 disclose use of an anisotropicmembrane, but they do not immobilize immunoactive materials within themembrane structure, an essential aspect of the present invention.Moreover, Breslau is not believed to teach a diffusion-only process butone wherein hydraulic pressure is used as a driving force to move massacross his membrane.

A need for an improved blood detoxification system has continued toexist. The present inventors have worked to provide such a system.

OTHER ART

Processes for detoxification of blood through ultrafiltration membraneshave been suggested in published art. One patent, that to Larsson et al(U.S. Pat. No. 4,361,484) suggests that such processes can be suitablyaccelerated by reliance on a pumping action wherein the blood isconstantly pushed into and pulled out of pores in the membrane. Thistype of mechanical action on blood platelets, resulting in constantchanges in velocity and pressure, tends to promote platelet aggregationand rupture and increases the possibility of intravascular coagulationwhen the plasma containing platelet components are returned to thecirculation. culation.

SUMMARY OF THE INVENTION

Therefore, it is a principal object of the present invention to providea relatively gentle blood-treating apparatus that minimizes damage tocirculating cells while selectively removing pathogenic agents,including antigens, antibodies and antibody complexes from thebloodstream.

It is a further object of the invention to provide improved means toimmobiize a biologically-selective agent, exterior to the lumen surfaceof a hollow fiber, which will remove antigens, antigen-antibodycomplexes, and other harmful materials from the bloodstream.

It is another object of the invention to provide a high concentrationgradient between said blood and the biologically-selective agent,thereby obtaining a faster reduction of toxins from the blood.

It is a further object of the invention to incorporate the uniqueselection diffusion properties of hollow fiber ultrafiltration membranesinto a compact, efficient, detoxification system which can be used inconjunction with instrumentation of the kind currently employed inhemodialysis by substituting the nonspecific hemodialysis membrane witha cartridge containing antigen-specific immunoabsorbents in combinationwith hollow-fiber membranes.

Other objects of the invention will be obvious to those skilled in theart on their reading of this application.

The above objects have been substantially achieved by the use ofbiospecific molecules, normally protein molecules called antibodies, butfor some purposes, antigen molecules, exterior to the lumen surface ofhollow fiber tubes. These biospecific molecules and the exterior of thetubes are also in a closed-volume system so that, steady-state, there isonly diffusion flow through the membrane.

The following specific embodiments of the invention are contemplated:

1. Immobilization of an antibody exterior to the lumen surface of thehollow fiber tube. In order to make the antibody spatially "available"for contact with the antigen, it is often desirable to have a molecularspacer segment forming means for spacing the antibody from the wall ofthe exterior porous side of the hollow fiber membrane. This generalarrangement is preferred when the molecular weight of the antigen islarge, e.g., 100,000 Daltons or higher in molecular weight. A convenientrange of interior surface area of tubes present in a single parallelgroup and mounted within a single cartridge is from about 0.1 to 5square meters. When the antigen, or toxic substance is of low molecularweight, as is the case with digoxin, quinidine, propoxyphene napsylate,barbiturates, salicylates, or theophylline, the hollow tube need only bepermeable to small or medium size molecules. When the toxic substance islarge, such as anti-insulin antibody, antihemophilic antibody or animmune complex such as those involved in rheumatoid arthritis,myasthenia gravis, glomerulonephritis, lupus erythematosus or otherimmune complex or autoimmune diseases, the pores of the lumen must be ofsufficient size to allow diffusion of the antibody or immune complex butsmall enough to prevent passage of the smallest blood cells, thethrombocytes or blood platelets. In most cases, it will be desirable tohave an interior diameter of the hollow fiber from 100 to 400micrometers and a tubular length of 7 to 50 cm. When a particularapplication requires, a six- or eight-carbon methylene group isconvenient as a spacer or "handle" between antibody and tube wall.

2.Coating of the exterior porous surface of the membrane with albumin.When the antigen to be removed is readily absorbed by albumin or whenthe immunologically active antibody is more readily chemically reactedwith albumin than with the material of the surface of the hollow fiber,the spacer molecule may be a protein such as albumin.

The outer surface of a membrane can be considered a relatively porousmaterial compared to that of the interior surface which is normally theeffective filter surface of an ultrafilter membrane of the asymmetric,sometimes called anisotropic, type. Thus, for example, the exterior,porous side of a hollow fiber membrane may be treated with a 17% humanalbumin solution in saline. The albumin will coat the surfaces withinthe porous zone of the membrane structure (i.e. the zone that underliesthe barrier layer of the membrane) and, thereafter, a solution ofprotein (antibody) can be deposited upon the albumin. Often it isdesirable to crosslink the protein somewhat (as with a diluteglutaraldehyde solution or some other such mild crosslink-inducingagent); this aids in anchoring the material in place on the hollow fibersurface.

When hollow fiber cartridges are prepared for the removal of substanceswith high albumin affinity, such as bilirubin, it is sufficient to"immobilize" high concentration of albumin on the outer, i.e. shell,side of the hollow fiber membranes with either of the methods describedabove.

It will be understood that, in the convention used in this Application,the troublesome blood component is called an antigen. In some cases thistroublesome component may be known to the art as an "antibody". In suchcases, it will be immobilized on a substrate which is immunoreactivewith it, for example an antigen or an antihuman antibody, or sequesteredin any of the other ways described herein as if it were an antibody.

SOME SPECIFIC USES FOR THE INVENTION

(1) Immobilization of serum albumin for the removal of excessmetabolites with high albumin affinity, such as bilirubin, for thetreatment of hyperbilirubinemia of hepatic origin in the neonate.

(2) Immobilization of specific antibodies for the removal of excessdrugs, intoxicants, or toxins. These monoclonal or polyclonal antibodiesare produced in cell culture systems or in animals according toprocedures known in the art. Such antibodies, or their Fab fragments,can be used, e.g. for digoxin or quinidine overdose during treatment ofcardiac patients or for accidental or intentional overdose ofsalicylates, theophyllines, analgesics, thyroxine-like compounds,barbiturates or tranquilizers, and for drugs of abuse.

(3) Immobilization of antibody directed toward a particular antigen, ortoward an activated immunoglobulin bound to the particular antigen. Forexample, in patients with systemic Lupus Erythematosus, rheumatoidarthritis, myasthenia gravis, and other such immune complex diseases,removal of circulating immunocomplexes which tend to cause multipleorgan damage may be of great benefit. Presently, plasmapheresis is beingemployed in a number of medical centers for the removal of circulatingimmunecomplexes.

(4) Immobilization of "antigen" to remove "antibodies" in thecirculation directed toward that antigen. One example of a disease inthis category is Hashimoto's thyroiditis. In this disease, circulatingantibodies against thyroglobulin deposit in the thyroid gland and causechronic goiter; removal of circulating anti-thyroid antibodies may beaccomplished with specific antihuman antibodies immobilized inmultitubular cartridges. Another example is a form of insulin-resistancediabetes. In this disease, high concentration of antibodies specific forinsulin circulate in the blood and neutralize insulin that isadministered. Selective removal of these antibodies using a boundinsulin antigen in the exterior lumen of the device can be used toreduce the plasma level of those undesirable antibodies.

(5) Immobilization of bioactive proteins with affinity toward pathogenicimmunoglobulins, e.g. immobilization of Protein A from Staphylococcusaureus for the removal of tumor associated blocking antibodies inpatients with colon carcinoma, melanoma, etc. These blocking antibodiesinterfere with the action of regular immunologic forces of the patientsagainst tumor cells.

Blocking antibodies were shown to be a particularly important feature incertain types of cancer. Removal of blocking antibodies frees the immunesystem to exert its antitumor activity.

ILLUSTRATIVE EMBODIMENTS OF THE INVENTION

In this Application and accompanying drawings there is shown anddescribed a preferred embodiment of the invention and suggested variousalternatives and modifications thereof, but it is to be understood thatthese are not intended to be exhaustive and that other changes andmodifications can be made within the scope of the invention. Thesesuggestions herein are selected and included for the purposes ofillustration in order that others skilled in the art will more fullyunderstand the invention and the principles thereof and will be able tomodify it and embody it in a variety of forms, each as may be bestsuited to the condition of a particular case.

IN THE DRAWINGS

FIG. 1 is a partially schematic view of an apparatus useful in practiceof the invention.

FIG. 2 is a section of FIG. 1.

FIG. 3 is a greatly enlarged cross-section of a hollow-fiber membranestructure constructed according to the invention.

Referring to FIG. 1, it is seen that a blood processing cartridge 10 isformed of an interior, blood-processing chamber 12 formed of interiorglass wall 14. Around chamber 12 is an optional exterior chamber 16which may be utilized to control the temperature in chamber 12; thetemperature of which is kept constant. A temperature-controlling fluidwould be passed into chamber 16 through lower conduit 18 and passes outof the chamber through upper conduit 20. Chamber 12 contains about fivehundred tubular, anistropic, ultrafiltration membranes 22 of the typegenerally called "hollow-fiber membrane". In fact, these membranes areabout 0.3 millimeters in inside diameter an 0.5 millimeters in outsidediameter. They have interior retentive membrane walls of about 1 micronin thickness and have a nominal central conduit path of about 8 inches.

The membranes are sealed together in matrices of resin 30 at the inletport 32 and outlet port 34. The resin effectively seals anycrossectional area which may be left between the membranes and assuresall blood entering the apparatus through inlet 32 flows into the tubularmembranes 22.

Immobolized on the exterior of membranes 22 is the antibody or othersuitable toxin-attractive material.

EXAMPLE 1 PREPARING ANTIBODIES FOR IMMOBILIZATION

In all the cases cited below, polymers are activated, i.e. madesusceptible for combining chemically with an antibody, by usingprocesses known in the art. Several useful polymers are cited, but sincethe polymer acts as a carrier rather than a physiological agent, anynumber of other polymers may be used as the substrata for carrying theantibodies. The primary requirement is that the antibody can conjugatethereto in a way which allows those molecular characteristics, whichgive the antibody its physiologically specific character, are leftspatially free to interact with an antigen.

(1-a) A soluble, or at least dispersible antibody is prepared by any ofthe number of methods including the following: An antibody suitable forany particular application is dissolved in a concentration of 50-200mg./ml. with human serum albumin in a phosphate-buffered aqueous mediumof pH 7.0, all according to procedures known in the art.

Gluteraldehyde in a concentration of 0.05-10% (depending on the protein)is added to the solution which is then incubated 12 hours at 4° C.Excess gluteraldehyde that remains in the reaction mixture isconveniently removed by addition of glycine to the solution at the endof incubation. This solution is then diafiltered through a membranehaving a minimal rententivity value of 50,000 molecular weight (50,000Daltons). The diafiltered antibody-bearing product is dissolved insaline or dialysis fluid.

(1-b) To obtain a reactive polymer, polyacrylic acid polymer (e.g. withmolecular weight of about 1,000,000) is activated by the azide procedure(Erlanger B C, Isambert M F, and Nichelson A M: Biochem.Biophys.Res.Commun.40, 70, 1970; Wilchek M, and Miron T Molecular &Cellular Biochem.4, 181, 1974). When the finely dispersed reactivepolymer is suspended in a buffered saline solution of the antibody ofchoice, the protein antibody will be bound to the polymer. It isimportant that the ratios of antibody to reactive polymer be selected toavoid excessive reaction which may result in an insoluble product. Ifthis ratio is appropriately adjusted, the spacing of the antibody alongthe polymer chain will allow a binding of the antibody with the antigenwithout untoward steric hindrances and the antibody conjugate remainssoluble.

(1-c) A copolymer of ethylene and maleic acid anhydride, for example ofmolecular weight 1,200,000, is partially hydrolyzed by incubating in achamber with controlled humidity and temperature and reacted with anantibody in e.g. saline solution. Then the resulting solubleprotein-polymer conjugate is used as a specific binding agent in asolution contained in the cylinder surrounding the outer walls ofhollow-fiber membranes.

(1-d) Dextran (Mw 10,000-40,000 Pharmacia Fine Chemicals, Piscataway,N.J.) is activated by the cyanogen bromide method (Axon et al: Nature214, 1302,1967) and reacted with the antibody in physiological salinemedia. The molecular weight of the conjugate obtained by this reactionis preferably higher than 1,000,000.

It is to be understood that when one activates a polymer, rather thanstarting with a pre-activated polymer of known molecular size, it ispreferable to filter the activated product to remove undesired lowmolecular weight material which may have been created during theactivation procedure.

EXAMPLE 2 SELECTIVE REMOVAL OF IMMUNOGLOBULIN G FROM BLOOD WITHIMMOBILIZED PROTEIN A

This process for purifying blood of a living animal by passing the bloodthrough an extracorporeal shunt device that includes an apparatus asdescribed herein for selectively absorbing undesirable substances fromthe blood can have many therapeutic uses including: (a) removal of redblood cell auto-antibody for treatment of lymphocytic leukemia andauto-immuno hemolytic anemia, (b) removal of tumor specific antigen fortreatment of colon carcinoma and melanoma, (c) removal of multiplemyeloma specific immunoglobulin G for relief of hyperviscosity inpatients with multiple myeloma, etc.

As known in the art, factor Protein A can be isolated from the cell wallof Staphylococcus aureus Cowan I (NCTC 8530) and the activity of theprotein is measured by immunodiffusion. The product so obtainedspecifically binds to the Fc portion of immunoglobulin G subclones 1, 2and 4 of man.

The cellulosic hollow fibers with an ID of 200 microns and inner wallthickness of 30 micrometers are potted to form a cartridge having atotal tube inner-surface area of 0.6 square meter. The inner walls arewashed with 0.2M sodium hydroxide solution, and after drying, areperfused with a 0.1M solution of bromoacetylchloride in acetonitrile andthe solution is recirculatd for 2 hours at room temperature. Thereafterthe inner walls are thoroughly washed with acetonitrile to remove excessreagent and dried in a nitrogen stream. Subsequently, the hollow fibersare perfused with a solution of Protein A in saline buffered with borateto maintain pH 8.5. After three hours of recirculation, the cartridge iswashed extensively with saline. The solution is assayed for Protein A byimmunodiffusion technique, known to the art, before and after therecirculation step in order to evaluate the degree of protein uptake bythe fiber wall. Finally, the cartridge is washed with a streptomycinsolution. Subsequently the cartridge is dried in a nitrogen stream,placed in a plastic bag and sealed.

Then, Protein A in a concentration of 50-200 mg/ml solution is incubatedwith a 0.05-10% glutaraldehyde solution in order to obtain soluble macromolecular aggregates having a molecular weight over 1,000,000. Afterremoval of small species by diafiltration, the super-Protein A moleculesare stored in saline solution at 4° C.

A cartridge containing 1,000 asymmetric hollow fibers having a cut-offpore-size at 500,000 Dalton MW and inner diameter of 150-200 microns iswashed by pumping saline solution through the fiber walls from theoutside in "reverse" ultrafiltration mode. Thereafter a 1% solution ofhuman serum albumin is pumped through the fiber wall in the same way inorder to deposit a monomolecular layer of albumin at the porous surfaceof the outer side (shell side) of the tubular membrane. Thereafter thesolution of super-Protein A is filtered through the walls of hollowfibers in "reverse" direction so that the crosslinked macromolecularaggregates of the biospecific binding agent are deposited directly atthe shell side of the membrane or in close promixity of the membrane inthe shell region. The activity of the solution before filtration andthat of the filtrate leaving the lumen is assayed for Protein A. Thebalance gives the amount of immunological activity deposited in thecartridge. After flushing the lumen space of the hollow fiber withstreptomycin and saline, the cartridge is placed into a plastic bag andsealed. Cartridges so prepared are used in extracorporeal shunts.

EXAMPLE 3

A specific antibody containing cartridge for use in the extracorporealshunt treatment of patients suffered from toxic levels of therapeuticdrugs was prepared:

Hollow fiber cartridges having lengths of 250 mm and containing 535hollow fibers were supplied by Amicon. The fiber dimensions are: I.D.180 micron and O.D. 360 micron, and the total contact surface area inthe cartridge is 750 cm².

The hollow fibers were first washed with 300 ml of distilled water inthe reversed ultrifiltration mode in order to remove glycerol used tomaintain wettability of the polysulfone hollow fiber membranes duringstorage and shipment. 500 mg. of human serum albumin (supplied by Sigma)are dissolved in 250 ml 0.01M phosphate buffer, pH 7. An aliquot of 50ml of albumin solution is pumped, in a reverse mode, through ports ofthe housing into what will be closed space around the hollow fibermembranes to deposit a thin albumin layer on the surface of the poroussponge structure surrounding each membrane. Thereafter a solution ofdigoxin antibody capable of binding 750 micrograms of digoxin is made in50 ml of 0.01M phosphate solution, pH 7, and loaded into the cartridgein the same way as the albumin in the previous step. Thereafter, 200 mlmore of the albumin solution is filtered into the outerspace of thehollow fibers.

Glutaraldehyde was dissolved in 0.01M phosphate buffer, pH 7.0, in orderto obtain 1% (w/v) solution. 25 ml of the glutaraldehyde solution waspumped into the extrafiber space of the cartridge and filtered throughthe fiber walls into the lumen over a period of ten minutes. Then 500 mlof saline was pumped through the cartridge in the reversedultrafiltration mode to remove unreacted glutaraldehyde.

After this treatment, the filtrate emerging from the lumen of the hollowfiber contained no traces of glutaraldehyde. The ports of the housing ofthe cartridge were closed with plexiglass disks and the lumen andlumen-access compartments were prepared by washing with one liter ofpyrogen free sterile saline solution. The cartridge, still wet with thesaline solution, was sealed into a plastic container and sterilized byexposure to a gas mixture containing 7% ethylene oxide in carbondioxide.

Sheep antidigoxin antibody, as described above, was immobilized withinthe cartridge and exterior to the interior lumen of the hollow fibermembranes. To 150 ml., of human blood was added 11,000 nanograms permillimeter of digoxin. After connecting the antibody containingcartridge to a fluid circulating system designed to permit passage ofblood into the center conduit of the multi-hollow fiber cartridge,digoxin containing blood was circulated through the system at a rate of100 ml. per minute. Samples of blood were removed for assay of digoxinat times 0, 5, 15, 30, 45 and 60 minutes after the start of thecirculation treatment. It was found that the concentration of digoxin inthe circulating human blood was significantly reduced after 5 and 15minutes of circulating through the system. After 60 minutes of treatmentof digoxin concentration of the blood was reduced from the initial11,000 nanograms per millimeter to 8,350 nanograms per millimeter whichcorresponds to a total of 400,000 nanograms of digoxin removed from the150 ml. of blood.

EXAMPLE 4

A drug specific antibody cartridge similar to that employed in Example 3(but immobilizing a quinidine antibody) for use in the treatment ofpatients suffering from toxic levels of therapeutic drugs was preparedand utilized in a manner similar to that described. To 200 ml of humanblood was added 35,000 nanograms of the cardiac drug quinidine. Theblood was circulated through the cartridge for one hour and samples ofthe blood were removed at intervals. The concentration of quinidine inthe blood was found to be: Initial concentration, 174 nanograms per ml.,5 minutes treatment sample, 153 nanograms per ml., 15 minutes treatmentsample, 153 nanograms per ml., 30 minutes treatment sample, and 148nanograms per ml. which corresponds to a total of 5,200 nanograms ofquinidine removed from the 200 ml. of blood.

EXAMPLE 5

A drug specific antibody cartridge similar to that employed in Example3, but proposed for treatment of patients suffering from toxic bloodlevels of drugs of abuse such as barbiturates, tranquilizers andanalgesics was prepared utilizing sheep anti-barbiturate antibodies. To200 ml. of human blood was added 8.8 mg. phenylbarbitol sodium. Aftertreatment of the blood and analyzing the blood, it was found that theconcentration of barbiturate in the blood at the various sample timeswas as follows: Initial, 44 micrograms per ml., and 60 minutes, 37micrograms per ml. Therefore the cartridge removed a total of 1,400micrograms of barbiturate from the 200 ml. of blood.

EXAMPLE 6

A drug specific antibody cartridge similar to that employed in Example3, but proposed for treatment of patients suffering from toxic bloodlevels of drugs of abuse was prepared utilizing antibodies to thepropoxyphene napsylate sold under the trade designation Darvon. To humanblood was added propoxyphene napsylate monohydrate. After treatment ofthe blood, it was found that the concentration (micrograms perdeciliter) of drug in the blood at the various sample times was afollows: Initial, 240, fifteen minutes, 90, thirty minutes, 10.

EXAMPLE 7

Examples of diseases characterized by excess antibodies orantigen-antibody complexes are lupus erythematosus, rheumatoidarthritis, myasthemia gravis, Factor VIII resistant hemophilia andinsulin resistant diabetes. To illustrate the use of this invention inthe reduction of blood antibodies and antigen-antibody complexes, acartridge was prepared as follows:

15 g silica gel (SMR-1-89C, Davison Chemical) having mean pore andparticle diameters of 300 A and 10 micron, respectively, were slurriedin 100 ml of 1M hydrochloric acid and after overnight stay filtered andwashed with distilled water until the filtrate was chloride free.Thereafter the silica was washed on the filter twice with 50 ml ofacetone, sucked dry and kept in the oven at 100° C. for 12 hours.

After the silica was cooled to room temperature, it was suspended in 60ml of toluene containing 7.5 g of gamma-aminopropyltrimethoxysilane(Petrarch) and under stirring the suspension was heated and kept onreflux over ten hours. After the suspension was cooled to roomtemperature it was filtered and washed with 50 ml of toluene, 50 ml ofmethanol and finally with 50 ml of acetone. The product was dried in theoven at 50° C. for three hours.

0.75 g of bovine insulin supplied by Sigma was dissolved in 7.5 ml of0.05M phosphoric acid. Then a concentrated sodium bicarbonate solutionwas added dropwise from a burette to the insulin solution under stirringand the pH was monitored. When the pH of the solution reached 4.5 thesolution became cloudy. The addition of sodium bicarbonate continueduntil pH of the insulin solution reached 6.6 and thereafter the solutionwas stirred for five hours until it became clear again.

The aminopropylsilica described above was suspended in 50 ml of 5% (w/v)glutaraldehyde solution in 0.01M phosphate buffer, pH 7.0, and stirredfor 20 minutes. Subsequently the suspension was filtered and washed withthe above phosphate buffer until no glutaraldehyde was found in thefiltrate.

The wet filtercake of the glutaraldehyde-treated aminopropylsilica wassuspended in the above described insulin solution and stirred for 40minutes. Therafter the slurry was filtered and washed extensively with750 ml 0.01M phosphate buffer, pH 7.0. The filtercake was resuspended in50 ml of phosphate buffer, stirred for 20 minutes and centrifuged. Thistreatment was repeated four times and testing of the last supernatantfor insulin was negative.

A hollow fiber cartridge sold under the designation "Plasmaflux P2" byFresenius was used which has the following specifications:

Membrane: polypropylene

Inner diameter 330 micron

Wall thickness: 150 micron

Contact surface area: 0.5 m²

Priming volume lumen: 52 ml

Extrafiber space: 195 ml

Housing material: SHN/PC

Potting material: Polyurethane

was washed with one liter of saline solution to remove the glycerol usedin maintaining the wettability of the asymmetric polysulfone hollowfibers. Thereafter the silica particles containing the immobilizedinsulin were dispersed in 500 ml saline solution and pumped into theextrafiber space of the cartridge which was gently shaken during thisprocess. The reversed ultrafiltration of the slurry resulted in adeposition of the siliceous affinity absorbent particles on the outershell of the hollow fibers. After a uniform distribution of theparticles was accomplished, first, 1.5 L of sterile, pyrogen-free salinesolution was filtered from the extrafiber space into the lumen of thehollow fibers, then 1.5 L of pyrogen-free sterile saline was pumpedthrough the lumen compartment.

Subsequently the ports of the housing of the cartridge were closed, thecartridge was sealed into a plastic container and sterilized by ethyleneoxide.

The cartridge is then connected to a circulatory system and used topurify blood from a diabetic patient with a high blood plasma insulinantibody titer. Such a treatment will cause the removal of importantquantities of the insulin antibody (an "antigen-antibody") from thepatient's blood.

In an experiment, where 93 ml blood, diluted with an anticoagulant, wasrecirculated through a hemopurifier with immobilized insulin, thefollowing changes were observed in insulin-antibody titer, as measuredby agglutination of insulin-coated sheep red cells by the tested serum:Initial titer: higher than 1:10 dilution at 30 minutes, the 1.4 dilutionwas positive, 1:5 was negative. Thus, the hemopurifier cartridge hadremoved 60% of the circulating insulin antibody.

EXAMPLE 8

An albumin containing cartridge was prepared as follows:

Hemoflow F-60 capillary dialyzer (obtained from the supplier, Freseneusof Germany) was used. The specifications of the hollow fiber cartridgesare as follows:

Membrane material: polysulfone

Housing material: polycarbonate

Potting compound: polyurethane

Net weight: 165 g

Inside diameter/wall thickness: 200/40 micron

Surface area: 1.25 m²

Priming volume blood: 75 ml

Priming volume dialysate: 230 ml

Flow resistance blood: 40 mm Hg

The protective glycerol coating of the fibers was removed by washing thecartridge with 1.5 liters of saline solution by passing the solutionthrough it as if it were to be ultrafiltered.

A quantity of 10 g of bovine serum albumin, Fraction V Powder (suppliedby Sigma), were dissolved in 1 Liter of 0.01M phosphate buffer, pH 7.0,and pumped through the two ports of the housing into the hollow fibercartridge in order to deposit the protein in the spongelike outer shellof the polysulfone fibers by a filtration in the reverse direction. Theflow rate was 10 ml per minute and slight vacuum was applied to bothends of the lumen compartment of the cartridge.

Glutaraldehyde (Fisher) was dissolved in 500 ml of 0.01M phosphatebuffer, pH 7, in order to obtain a 1% (w/v) glutaraldehyde solution.

After the albumin solution was filtered into the hollow fibers theglutaraldehyde solution was pumped into the cartridge in the same way.The flow rate was 15 ml/minute. Upon contact with the glutaraldehyde,the bovine serum albumin deposited at the outer surface of the capillarymembrane proper in the porous shell became crosslinked and therebyimmobilized at the polysulfone surface.

The cartridge was washed with 1.5 L of pyrogen-free and sterile salinesolution, first in the reversed ultrafiltration mode, then through thelumen compartment. Thereafter, the cartridge with hollow fibers stillwetted by saline use was sealed into a plastic container and sterilizedby ethylene oxide.

The albumin cartridge was connected to a circulating system and 100 mlof blood obtained from a patient suffering from hyperbilirubinemia waspumped through the system. The initial concentration of totalbilirubinemia in the blood was 29 milligrams per deciliter blood. Aftertreatment for 100 minutes the concentration of bilirubinemia haddecreased to 19 milligrams per deciliter which corresponds to a total of10 milligrams of bilirubin removed from the 100 ml of blood.

EXAMPLE 9

A column was prepared, according to the procedure of Example 3 but usinga specific antibody to 1-thyroxine as the immunoreactive agent on theexterior side of a membrane device as described above. The membraneswere of the nominal 10,000-Dalton cutoff type. Into a quantity of 175 mlof human blood, was added 72 micrograms of Na 1-thyroxine. The initialconcentration in the blood was 0.41 micrograms per milliliter. After 60minutes of treatment, the concentration had dropped to 0.348 microgramsper ml. Thus 11 micrograms of Na 1-thyroxine was removed from the 175 mlof blood.

In appraising the advantages of the invention, it appears that theparticularly beneficial properties are achieved by providing asteady-state diffusion process across the wall of the membrane whereby ahigh concentration gradient is maintained across the membrane by theplacement of the antigen-attracting material deposited on the exteriorof the membrane. Because the volume of the container of the membranes isboth closed and of very limited volume, there are only trivialquantities of relatively small blood chemicals lost during the start-upof operation of the cartridge. Thereupon, a diffusion equilibrium(steady-state) is established with respect to such chemicals and nosubstantial further net loss of such chemicals occurs excepting, ofcourse, the antigens rejected by the immunoabsorbent.

It will be apparent that the optimum size of the membrane pores willdiffer from application-to-application, but in no event will they belarge enough to admit the formed elements of the blood. Hydraulic flow,as opposed to diffusive flow, does not take place at all oncesteady-state operation is established. In particular, the push-pullaction of blood components in the pores of the membrane (as proposed byLarsson et al in U.S. Pat. No. 4,361,484) is wholly avoided because theclosed, constant-volume container for the membranes permits nofunctional hydraulic pulse to be transferred therethrough.

It is also to be noted that bacteria can also be attracted across themembrane and immobilized outside the blood stream.

The antibody can be linked to a spacer molecule. Such spacers areusually the length of several carbon atoms, say up to about 20 but oftenabout 4 to 12 carbons in length.

Depending on the nature of the antigen to be removed, the nominal poresize can be quite large, but never large enough to admit blood cells.Nominal pore sizes (globular-molecule separation test known in the art)of 50,000 are effective in the antibody applications disclosed hereinbut antigen-antibody-type toxins require pore sizes that are up to about500 millimicrons in nominal diameter. For some smaller antibodies, apore size of only about 10,000 Daltons is acceptable. In the preferredembodiments of the invention, not only will blood cells be preventedfrom passing through the membrane, but all formed elements of the blood,i.e. the erythrocytes, leukocytes, and thrombocytes, will be maintainedexclusively on the barrier side of the membrane.

Mixtures of antibodies can usually be used in a substantially additiveway. In such a case the removal of a plurality of antigens appears to beessentially non-competitive.

Albumin, besides being an easily-immobilized collector of some toxinsfrom the blood, is also a good material by which to bind or immobilizeother antibodies. For example antidigoxin antibodies can be bound tosuch blood. Sometimes it is desirable to polymerize antibodies forimmuno-scavenging use. Antidigoxins is an example of this. By doing so,one can allow use of larger effective diffusion pores in the membranewithout danger of back diffusion of the immunoscavenging means.

It is also to be understood that the following claims are intended tocover all of the generic and specific features of the invention hereindescribed and all statements of the scope of the invention which mightbe said to fall therebetween.

What is claimed is:
 1. Apparatus for removing selected antigens from aphysiological fluid comprising an anisotropic membrane surface of thetype having a retentive skin side and a porous substrate exterior sideand forming a diffusion barrier permeable to water and said selectedantigens but substantially impermeable to formed elements of blood, aflow path forming means for directing said fluid to flow in contact withthe retentive skin side of said membrane; pump means to move said bloodalong said flow-path, said pump means being the only blood-moving meansof said apparatus; a closed, constant-volume compartment enclosing theexterior of said membranes; and an immunoreactive scavenging agenteffective to scavenge predetermined antigens from said blood immobilizedon the porous substrate side of said anisotropic membrane.
 2. Apparatusas defined in claim 1 wherein said membrane barrier surface is in theform of the interior surfaces of a plurality of closely spaced tubes andwherein said agent is positioned within the porous exterior portion ofsaid tubes.
 3. Apparatus as defined in claim 2 wherein said antigen isdigoxin.
 4. Apparatus as defined in claim 2 wherein said antigen is abarbiturate.
 5. Apparatus as defined in claim 2 wherein said antigen isa propoxyphene napsylate.
 6. Apparatus as defined in claim 2 whereinsaid antigen is an antigen-antibody complex.
 7. Apparatus as defined inclaim 6 wherein said antigen-antibody complex is an insulin antibody. 8.Apparatus as defined in claim 2 wherein said antigen is quinidine. 9.Apparatus as defined in claim 2 wherein said antigen is bilirubin andsaid antibody is albumin.
 10. Apparatus as defined in claim 2 whereinsaid antigen is immunoglobulin G.
 11. Apparatus as defined in claim 2wherein said antigen is a bacteria.
 12. Apparatus as defined in claim 2wherein said antigen is 1-thyroxin.
 13. Apparatus as defined in claim 1comprising additionally an exterior chamber around said constant volumecompartment forming means to confine a temperature-control fluid tomaintain said compartment at constant volume.
 14. Apparatus as definedin claim 13 wherein said membrane barrier surface is in the form of theinterior surfaces of a plurality of closely spaced tubes and whereinsaid agent is positioned within the porous exterior portion of saidtubes.
 15. Apparatus as defined in claim 1 wherein said antigen is about100,000 Daltons and wherein said immunoreactive scavenging agent isattached to said membrane via a molecular space segment.
 16. Apparatusas defined in claim 15 wherein said spacer material is formed ofalbumin.
 17. Apparatus as defined in claim 15 wherein said immobilizedscavenging agent is albumin.
 18. Apparatus as defined in claim 1 whereinsaid immunoreactive scavenging agent is in the form of an antibody whichis chemically reacted to a high-molecular weight substrate to inhibitdiffusion from said porous substrate of said membrane.
 19. Apparatus asdefined in claim 18 wherein said high molecular weight substrate issilica gel or dextran or a polymer formed of said scavenging agent. 20.Apparatus as defined in claim 18 wherein said immobilized scavengingagent is albumin.